JPS6184561A - Immunoassay system which do not depend upon antigen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Honjuku Sarashi! The present invention relates to an immunoassay system that uses anti-idiotypic antibodies instead of antigens and is therefore antigen-independent. The invention also relates to methods for producing anti-idiotypic antigens.

Competitive immunoassays rely on free antigen in the sample.
It has long been used to determine the presence of antigen in a sample by measuring the inhibition of standard antigen-antibody complex formation, typically bound on one side and labeled on the other. In addition, typical quantitative immunoassay kits include standardization samples of pure antigen, so that a reference solution can be run in parallel with the sample to minimize sampling error and ensure accuracy. There is. In many cases, antigens are extremely difficult to release and purify, and may be of limited stability.

Antibodies that recognize highly variable or idiotypic regions of an antigen are involved in regulating the immune system. They are known as anti-idiotypes (a-Id). Thus, an antibody that specifically binds a particular antigen (a-Ag) also specifically binds its complementary a-Id. The antigen in the sample is labeled a-Ag.
It has been suggested that complementary a-Ag/a Id antibody pairs can be used in competitive immunoassays where the extent of a-Id/aAg complex formation is inversely proportional to the antigen concentration in the sample. ing.

For example, Potocnjak et aL, 5cie
nce+ 215+ 1637 (1982);
Mitchell et al.
, J, ux, Bio1Med Engineering” play,
See 11.27 (1983). However, a
There is no suggestion of using a-id as a replacement for S antigens.

Typically, a-Id administers a-Ag in an antigenically active form to an animal, challenges it, and collects antiserum, or collects lymphocytes and fuses them with myeloma cells to generate hybridomas. It is produced by creating. a
There is no suggestion of using lymphocytes from animals with growing hybridoma tumors and secreting monoclonal a-Ag as a source of -Id.

One object of the present invention is to provide a competitive immunoassay method that does not rely on the need for purified antigen in its routine use.

Another object of the present invention is to provide a method for producing anti-idiotypic antibodies that is simpler and more advantageous than conventional methods.

Upon further consideration of this specification and claims,
Still other objects and benefits of the invention will be apparent to those skilled in the art.

The purpose of these studies is to provide a method for determining the presence of an antigen (Ag) in a liquid sample, comprising: (al) the liquid sample and the shrimp on the antigen;
An antibody (a-Ag) that specifically binds to the a-A
incubate in the presence of a given amount of anti-idiotypic antibody (a-Id) that specifically binds an epitope on the hypervariable region of g, and determine the extent of a-Ag-a-Id complex formation. (b) using a standard solution of unlabeled a-Id in step (a), in which at least one of a-Ag and a-Id is labeled a'-Ag and a-Id labeled as such, and a-Ag
- measuring a standard value of the degree of a-Id complex formation, thereby
- By providing a method as described above, characterized in that the presence of said antigen is determined from the relative degree of a-Id complex formation.

The present invention further provides the steps of: (a) fusing lymphocytes from an animal growing an algal tumor capable of secreting idiotypic antibodies with myeloma cells; isolating and cloning the resulting hybridoma that secretes a monoclonal antibody that specifically binds the epitope.
A method for producing a monoclonal anti-idiotypic antibody is provided.

Competitive immunoassay, e.g. radioimmunoassay
(RIA), enzyme-linked immunosorbant assay
(ELISA) and the like are routinely used to detect and quantify the presence of antigen in a sample by measuring the extent of inhibition of the antigen/antibody reaction. Typically, either the antigen or the antibody is bound to a solid support, and the other member of the pair is labeled in some way to make it detectable. Such labels often include, for example, radioisotopes, enzymes, fluorescent markers, etc., all of which are well known to those skilled in the art.

A label may be attached to the component directly or indirectly, eg, by attaching the label to another molecule capable of recognizing one component of an antigen/antibody pair.

For example, an antibody can be indirectly labeled by coupling an enzyme, fluorescent marker, or radioisotope to an isotype-specific antibody that recognizes a nonvariable region of an antigen-specific antibody. Alternatively, a label can be coupled to an antibody that recognizes an available epitope of the antigen after binding of the antigen to a specific antibody.

It will be appreciated that many other variations of this broad concept are possible and known to those skilled in the art.

Numerous benefits are realized by substituting anti-idiotypic antibodies for antigen in competitive immunoassays. However, prior art suggestions for such substitution included substituting labeled a-Id for labeled antigen in the main competition reaction of the assay. Unlabeled antigen was always used to test inhibition. However, in routine assays, the need for purified antigen in any respect is a disadvantage. Antigens are often difficult to isolate and purify, and often have limited stability. The present invention provides the further benefit that reference measurements are performed using unlabeled a-Id, making the entire routine measurement antigen independent.

Characterization of the relative efficacy of antigen and a-Id as inhibitors of the a-Ag/a-Id complex must first be performed, but the homogeneity and stability of antibodies, particularly monoclonal antibodies, must be carried out first. It will be appreciated that this eliminates the need for continuous calibration and/or standardization of a-Id.

Although some embodiments of the method of the invention are appropriate, the invention is not limited to the embodiments specifically described herein, as alternative embodiments will be readily apparent to those skilled in the art. These illustrative examples will now be described in detail.

In a first preferred embodiment, an antibody that specifically binds the antigen to be measured, preferably a monoclonal antibody with high specificity for the antigen of interest, is bound to a solid support to facilitate separation. Will. This can be done in a number of ways, e.g. by treating a polyvinyl chloride test tube with a-Ag, introducing a given amount of radiolabeled a-Id with a measured volume of sample, and - carried out 11 by determining the degree of inhibition of binding to Ag-coated test tubes.

Reference measurements are performed using unlabeled a-Td whose ability to block aAg/a-Id complex formation has been previously quantified and correlated with known amounts of antigen.

Another preferred embodiment involves binding the a-Id to a solid support and labeling the specific a-Ag. again,
The concentration of antigen in the sample will inhibit binding of the labeled component to the solid phase, and the degree of inhibition will be correlated with a standard curve using rice Qa-Id and labeled a-Ag.

The term "antibody" as used herein includes all idiotypes, eg, IgM, IgG, IgA.

IgD and IgE, their isotypes such as Ig
It will be understood to include Cl, IgC, 2, etc., and fragments thereof such as F (ab')z, Fab, Fab', etc. a-Ag and a-I
One or both of d may be a whole antibody or an antibody fragment.

The methods of the invention are particularly useful in competitive immunoassays for tumor-associated antigens. various types of tumors and/or
Antibodies, particularly monoclonal antibodies, against antigens produced or associated with cancer-related pathologies have recently been developed. A number of such antibodies that are also useful for radioimmunodetection of cancer are described in particular in US Pat. No. 4,348,376.4.
, 362 , 544.4, 331° 647.4, 46
8,457.4,444,744.4.460.559
and 4,460,561. In addition,
Other antibodies are disclosed in US patent applications 609,607, filed May 14, 1984, and 633.999, filed July 24, 1984. The disclosures of the foregoing patents and patent applications are hereby incorporated by reference in their entirety. Antigens associated with various infectious lesions can also be detected by the method of the invention, and antibodies against such antigens are particularly disclosed in the aforementioned US Patent Application No. 633.999.

As mentioned above, a-Id is conventionally produced by challenging animals with an immunologically active form of a specific a-Ag. In some cases, this can be accomplished simply by injecting the appropriate animal, such as a mouse, sheep, goat, rabbit, etc., with the antibody in a conventional adjuvant. Booster shots of antibodies are administered periodically, and antisera are collected after a moderately hyperimmune state is achieved.

Antibodies can be recovered from antiserum by conventional purification procedures, including affinity chromatography on columns to which +0M antigen is bound. Cross-reactive antibodies can be achieved by chromatography on columns coupled with other cross-reactive antigens.

Alternatively, conventional techniques for producing monoclonal antibodies can be used. This involves challenging a suitable animal, e.g. a mouse, with an immunologically active form of a specific antibody;
It involves removing the lymphocytes, eg splenocytes, after a suitable high immune response has been obtained and fusing the splenocytes with a suitable myeloma cell line. Hybridoma clones are routinely produced and screened for specific anti-idiotype antibodies.

Human hybridomas are described in US Pat. No. 4,464,465 and Kosbor et al., 'The p
production of mono-clonal
antibodies from human lym
phocytes''.

Immunol, Today 4: 72-79.
1983; Kozbor et al.
man hydromas construct
d with antigen-specific E
pstein-Barr virus-transfo
rmed cells + Proc, N
ATL, Acad, Sci, 79:665
1-6655+ 1982; Kozber et
al,,” In vitro stimulus-I
Lymphocytes as a 5ou
race of human hybrid-domas
”, Eur, J., Iamunol, 1984
; (11th son etat,, ' Huma
n hybridoa + as producing m
onoclonalantibodies of pr
edefined antibiotic 5pecif
i-city”, Proc Natl, Acad
, Sci, (USA) 77:5429-5
434.1980; Croce et al.
, ”Production of human hy
bridomas secreting antibo
dy t.

measl es virus”, Nature
288:488-489.1980. All of the above is incorporated herein by reference.

These references disclose methods for producing human monoclonal antibodies. In order to adapt these methods to the production of human hybridomas secreting a-Id, the first step is to treat patients whose immune systems have produced antibodies against it, e.g. with certain tumors and/or infected lesions. The process involves collecting peripheral blood from the body. The antibody must have an a-Id to idiotype a-Ag.

The lymphocytes are then separated, for example on a Ficoll gradient,
It will be collected. Alternatively, splenocytes and/or lymphocytes can be removed from the patient's lungs and/or lymph glands or vessels. For a-Id production, lymphocytes e.g. by a-Id and/or Ag/a-
Tested by competitive inhibition of Ag complex formation.

The lymphocytes are then fused with human myeloma cells, such as the LICR human myeloma cell line, and the hybridoma cells producing a-Id are harvested and cloned. Instead, the lymphocytes are Epstein-Bar
r virus (EBV) and cloned to select clones that produce a-Id. EB
V-immortalized cells can also be fused with human myeloma cells, and a-Id-producing hybridomas can then be isolated and cloned.

Finally, isolated lymphocytes can be sensitized and stimulated in vitro with e.g. , the cells are then fused and/or immortalized as described above.

A preferred alternative method for producing monoclonal a-Id has been developed by the inventors. The method uses an isogenic animal in which the hybridoma cells are isogenic.

Most cases arise from an alternative method of manufacturing monoclonal antibodies in which mice are injected and one or more ascites tumors are grown in the appropriate part of the animal. After that, ascites fluid is removed and monoclonal antibodies are easily isolated from it.

Surprisingly and unexpectedly, lymphocytes excised from animals growing ascites hybridomas.

In particular, it has recently become clear that splenocytes can fuse with myeloma cells to form hybridomas that secrete anti-idiotype antibodies directed against antibodies secreted by ascites tumors. Thus, a convenient method is provided in which both components of the a-Ag/a-Id pair can be produced. This method can be used to produce a-Id clones that specifically bind antibodies secreted by any hybridoma that can pass through the host animal. This hybridoma has ascites 1! It can be passed as a tumor, subcutaneous tumor, or any other type of live, growing hybridoma that can secrete a-Ag.

A further improvement of this method is obtained by in vitro stimulation of anti-idiotypic clones. This is obtained, for example, by the addition of ascitic fluid to splenocytes isolated from an animal in which ascites has been grown, as well as the presence of a stimulant for B cell production. Suitable such stimulants are known and include, for example, burdock mitogen. This in vitro stimulation is preferably performed in tissue culture medium. The effect is to selectively focus and stimulate the growth of B cells that produce anti-idiotypic antibodies. obtained stimulation,
The cultured cells are fused with myeloma cells and an increased yield of a-Id hybridomas is observed.

Anti-idiotypic monoclonal antibodies offer technical and commercial advantages over conventional antigens in competitive binding assays. a-Id obtained from hybridoma
can be manufactured in unlimited quantities with limited specificity and high stability.

Hybridoma cell lines producing a-Id can be frozen in liquid nitrogen and stored indefinitely.

a-Id offers unique opportunities for biological and chemical modifications intended to improve the performance characteristics of test kits. For example, a-Id antibody fragments can be used that can offer advantages over whole immunoglobulins. For example, the bifunctional hybrids disclosed in US Pat. No. 4,33L647 or similar bispecific species can be used. Isotype switching can be performed with a-Id to realize the benefits and/or allow the use of a standardization or universal label attached to an intib-specific antibody.

In addition, immunoglobulin labeling techniques are highly developed and can be easily standardized.
It will often be easier to attach to a label and/or solid support than the antigen.

The ease of production and purification of a-Id monoclonal antibodies from tissue culture media or ascites fluid also favors their use. Furthermore, purified a-Id monoclonal antibodies are often cheaper to produce than purified antigens, especially in the case of tumor markers.

The specificity of single-a-Id is too narrow and the a-fi of the antigen.

If binding to a-Ag cannot be completely blocked, two or more a-Ids can be selected from a library with partitioned blocking properties for a-Ag in order to obtain adequate blocking. As mentioned above, a-Ag or a of the complex
Immunoglobulin fragments can also be used as the -Id component and/or as the a-Id used to generate standard curves.

As an illustration of the method of manufacturing a-Id and the construction and use of the assay kit according to the present invention, the following preferred embodiments are described in detail. The antibody used as the antigen to stimulate the production of a-Id was a purified anti-cancer protein designated NP-2, the manufacture of which is described in U.S. Pat. No. 609,607, filed May 14, 1984. It is a fetal antigen monoclonal antibody. This antibody was prepared in phosphate buffered saline (
PBS.

pH 7,2') at a concentration of 1 μ/ml and stored at -20° C. until immunization for assay.

Hibrotocol NP-2 for hybridomabis
Two immunization schedules are used to generate anti-idiotypes. Ba1b/cJ female mice, 6 to 8 weeks old (Jackson Laboratories).

Purified NP-2100mc in complete Freund's adjuvant (1:1) to Bar IIarbor+ ME)
g is injected intraperitoneally (i,p,). Animals were PB after 1 week.
given a second i,p injection of 50 mcg NP-2 in S, followed by 50 mcg NP-2 in PBS at 8 weeks.
receive a final i,p injection of. Animals are sacrificed for fusion three days after the final immunization.

An alternative immunization protocol involves isolating anti-NP-2 activated tiles from animals injected with the parental hybridoma cell line PL-A6. In this operation, female Ba1
b/cJ mouse is 0.5d prestar: / (2,6,1
0,14-tetramethylpentadecane; Aldrich
Chemical Co,, Milwaukee,
primed with an injection of Wl). 1-2 animals
X106 (7) PL-A6 cells are inoculated. Mice usually develop ascites tumors after 7-14 days, and ascites is collected by i.p. puncture. Animals are sacrificed for the 211th time and splenocytes are removed for the fusion step. Preliminary observations indicate that these animals develop an immune response to the idiotype.

51 Operations for hybridoma production are described by Kennette
tal,, Curr, Topics Micr
obiol, Immunol, +81+77 (
1978) ; 5trike et al, +
J, Immunol,, Nikimata.

179B (1984). The immunized animals were sacrificed by cervical dislocation, the lungs were aseptically removed, and the lungs were cultured in serum-incompatible Dulbecco's modified Eagle's medium (DMuM; Gibco Laboratories).
atories + Grand l5land + NY)
Tear to cleave the single cell suspension of splenocytes inside.

Cells are collected by centrifugation and washed once in DMEM. Total cell number is determined by hemocytometer counting using trypan blue exclusion technique.

Approximately 108 splenocytes are 2.5X107 P3X63-
AC8,653 murine myeloma cells (653, ATC
C). The supernatant is removed and the pellet is resuspended by gentle puncture.

Polyethylene glycol (PEG 4000; Gib
co Laboratories) 1 rtdl
is added to the tube and the cells are incubated for 90 seconds at room temperature. DMuM5 is added for 60 seconds, followed by DMEMS formulation supplemented with 20% fetal calf serum (D-20) for 60 seconds. The fused cells are then centrifuged at 500Xg for 10 minutes. The final cell pellet was 1:I D-20+HAT (D-
HAT) (HAT: Hypoxanthene-Aminopterin-Thymidine) was resuspended in 50 d of D-20 containing HAT (hypoxanthene-aminopterin-thymidine) and transferred into six 96-well microliter plates, approximately 2.3X10 per well. ' added at a concentration of '. Cells are 1, 3. D-HAT is fed on days 7 and 10 and D-20 weekly thereafter until colonies appear and are assayed (approximately 2-3 weeks).

Initial screening for the presence of NP-2a-Id is performed using RIA using NP-2 monoclonal antibody (MAB) as the coating antigen. PVC microtiter plate is 0.05
Goat anti-mouse Fc (Pel Freeze) suspended in M carbonate buffer (pH 9,6).

Rogers + ^R) at 1 mcg/well.

1-2 mcg of NP-2 is added to each well over a period of 1 hour, followed by 50 μ2 of hyperidoma supernatant. L25I-labeled CEA was added and each well was analyzed using a Puncard autogamma scintillation spectrometer (
Model 5230''). Positive colonies are identified by inhibition of CEA binding to NP-'2.

After freezing aliquots of these positive colonies at -135°C, large numbers of hybridomas are cloned by limiting dilution. Systematic dilutions of positive colonies are made up to a final concentration of 10 cells. 10
0 μl aliquots are added to each well of a 96-well (hard-well) microlin plate containing a feeder layer of isogenic splenocytes. Macroscopic colonies are observed in approximately 10 to 21 days. Only containing wells (visible macroscopically) are reassayed in the RIA described above.

Positive clones are removed and expanded in tissue culture. Clones are frozen for future use and cell 0
．． Subcloned to manageable numbers by limiting dilution at a concentration of 5 to 11 cells/well. Subcloning is a competitive radioimmunoassay (RIA)
are tested for their effect on CEA binding to NP-2 using

It is necessary to determine the effect of a-Id monoclonal antibody concentration on the binding of CEA to a-Id'l .1 Ru-dioimmunoaseth NP-2. This is implemented using competitive RIA. A series of test tubes is prepared and each person is given 0. (11 M ammonium acetate and 20 d of NP-2 (1:100) are added. Varying concentrations of a-Id plus l-125 labeled CE
50 μm (approximately 10' cpm/m) of A is added and the tubes are incubated at 45° C. for 30 minutes.

During each incubation period of 41 t, two doses of Z-gel (zirconyl phosphate) were added and 10
Centrifuge for 5 minutes at 00xg. The gel is washed once with ammonium acetate and the tubes are counted in a gamma counter.

Appropriate positive and negative control tests (including inappropriate MAB) are performed to ensure accuracy of results. NP
- a-I that results in blocking the binding of CEA to 2MAB
Only the d subclones are preserved. It is expected that a large number of subclones will exhibit blocking activity. These cells are expanded by tissue culture and frozen at -135°C. Large-scale production of the desired MAB i) in ascites from Ba1b/cJ mice! Will include iIM.

Monochrome-containing 7-1 hybridomas are grown in DMEM supplemented with 10% fetal bovine serum. Cells are spun down at 500xg for 10 minutes at 4°C and resuspended in serum-incompatible medium. Cell density is determined by counting suspended cells in a hemocytometer and viability is determined by trivan blue exclusion.

Balb/cJ mice, 8-1θ weeks old, are primed by intraperitoneal injection of pristane 1 3 to 4 days before inoculation. Inoculation is achieved by intraperitoneal injection of 1-2X106 live cells. To obtain ascites, mice were placed on metofane (Pitman-Moore).

Inc., Washington Crossing
, NJ) and puncture the ascites into a sterile camped plastic culture chamber with a 20 gauge hypodermic needle.

To remove cells, ascites was incubated at 500Xg for 15 minutes.
Centrifuged at <RTIgt;C,</RTI> the supernatant containing ascitic fluid is transferred to a sterile container and stored frozen at -20C to -30'C until required.

a-IdMAyB iso Ibhi anti-NP-2a-Id mouse antibody isotype was affinity purified heavyweight specific anti-mouse 1g (cappe
l Laboratories, Malvern+
PA) and in a modified EL ISA using a-Ids as the coating antigen. Anti-idiotypic IgG monoclonal antibodies are purified as described below.

The purification of these monoclonal antibodies consists of three main steps:
1) ammonium sulfate fractionation of crude ascites to obtain crude gamma globulin; (ion exchange chromatography (IEG) on 21D E A E-cellulose; and (3) chromatography on a hydroxyapatite column and linear potassium phosphate final purification of the IgG fraction by gradient development.

Plasma ■ Thaw the ascites, pool if necessary, and
Centrifuge at 0Xg for 15 minutes at ambient temperature and pass the supernatant through glass wool. An aliquot of a saturated solution of (NH4)zsO4 was diluted with concentrated ammonium hydroxide (30%) to pH 7.
Adjust to 5. Using a buret, (N) + 4) 2
A saturated solution of SO4 is added dropwise to the ascites with stirring to a final concentration of 40% saturation. After addition, the suspension is stirred at room temperature for 120 minutes. 1. Transfer the suspension to a capped centrifuge tube and collect the precipitate by centrifuging at 1800×g for 15 minutes at room temperature. Discard the supernatant and dissolve the pellet in a small volume of PBS. The dissolved pellets were added each time to 1000 volumes of O, OIM potassium phosphate.
Dialyze at 4°C against 6 exchanges of H8,0 (
(changed 3 times a day, every 3 to 4 hours). The turbidity formed during dialysis is removed by centrifugation at 1800×g for 15 minutes at room temperature.

75g of D E 52 (Whatman+ C11f
ton, NJ) is equilibrated with 0.5 M sodium phosphate pH 8.0 at room temperature with occasional stirring. This amount of ion-exchanged cellulose (IEC) is
It is sufficient to process material obtained from 00 to 120d. After 2 to 3 hours, reduce the IEC to 0. OIM Sodium Phosphate pH 8,0 p) of effluent and pressurized liquid
and conductive dust until they are equalized. IEC is at least 0. Washed with 1 bed volume of OIM potassium phosphate p 8.0.

Load a 1 g sample onto the column and chromatograph until the absorbance at 28Q nm of the effluent returns to baseline, change the buffer to 0.025M sodium phosphate pH 8.0, and continue the f4 separation at the same flow rate. The protein fraction that elutes at this phosphate concentration contains mouse IgG, and it shows IgG and small amounts of other proteins (possibly transferrin) that show migration on immunoelectrophoresis.
It becomes more.

This contaminant is easily removed by chromatography on hydroxylapatite. If necessary, the IgG fraction was concentrated by ultrafiltration using an Amicon YM-30 membrane and filtered with OIM potassium phosphate pH 6.8 containing 0.02% sodium azide for 2000 ml each time. Dialyze with BiC 4 times. Immunoelectrophoresis and PA
Samples are taken for analysis by GE.

Hydroxyl abate chromatography boots - IEC
1 g of hydroxylapatite (HA; biorad, Rict+mo) per purified IgG20
nd, CΔ) in 6 volumes (form/g) of 0. (Dispersed in 11 M potassium phosphate pH 6.8. Place the HA bed on approximately 0.5 cm of Sephadex G-25 fines (Pharmacia) in a thick short column (2.5 x 20 cm). . Both the Sephadex layer and HA are packed at a flow rate of 12-h r'X cm-2.I
[G fraction was centrifuged in isooxg for 15 min at room temperature to remove particulate matter), then at a reduced flow rate of 6 hr
' Apply to the column with X cn-2. Column with at least 1 bed volume of heavy liquid or “El” of effluent
Elute until the yield (280 nm) is at baseline.

Connect the column to a gradient former and add 0.0% of potassium phosphate. (Line gradient from 11 M to 0.03 M (
The total volume of the gradient is equal to 10 bed volumes) and i gtt
do.

The absorption of the effluent is followed at 280 nm and the IgG fraction is collected in a sterile pyrogen-free container. The IgG formulation is concentrated to 10 nw/m by ultrafiltration using an Amicon YM-3 (111A) and stored aseptically at 4°C.

This monoclonal antibody-type agent was tested for purity and immunoreactivity by (immunoelectrophoresis against all whole serum and anti-mouse IgG; (b) polyacrylamide gel electrophoresis in nondenaturing gels; or (c1 The 1gGg agent has a specific activity of 12-15μCi/mc at I251.
g and analyzed for immunoreactivity and purity using the following affinity matrix. CEA bound to Sepharose and anti-mouse IgG bound to Sepharose, the former to test for immunoreactivity, and the latter to test the percentage of IgG in the formulation.

In general, this kit requires that a standard concentration of radiolabeled anti-idiotypic monoclonal antibody and a patient's blood tn be
EA coated polyvinyl chloride test tube (a-CEA-P
VC). The concentration of CEA in serum is derived from the standard curve by correlating the inhibition of anti-idiotypic binding to a-CEA-PVC by patient serum.

1. a-CEA-PVC 8.1 Polyvinyl chloride test tubes are coated with anti-CEA monoclonal antibody suspended in carbonate buffer pH 9.6. Unreacted sites on the test tube (this can result in non-specific binding)
is blocked with 3% bovine serum albumin in the same buffer. These test tubes can be stored for 6-12 months at 4°C (Kennet, R, 11,.

Monoclonal Antibodies;
R, I (, Kennel, T, J.

McKearn+ K, B, Bechtol+ ed.
s,, Clenum Press.

N, Y., p. 376, 1981).

It is necessary to determine the concentration of a-Id monoclonal antibody that completely blocks the binding of CEA to the anti-CEA monoclonal antibody. To accomplish this aspect of test kit development, varying concentrations of 125I-labeled a-Id monoclonal antibody were combined with known concentrations of CEA in a-CEA-PV.
Add to C. An inhibition curve is obtained that relates the inhibitory activity of a-Id to CEA protein concentration. The a-Id is then used to obtain a standard curve in a diagnostic test kit.

3-MU-Stock This test kit from Eiip Monochrome is used to detect a-Id in human serum.
Takes advantage of the ability to act as a surrogate antigen for concentration measurements.

The following components must be supplied in the kit:

a-CEA-PVC0 conventionally labeled, e.g., by the chloramine T method, an IB-labeled anti-idiotypic monoclonal antibody prepared as described above.

Unlabeled a-IdMAB (for standard inhibition curve generation).

Phosphate buffered saline (PBC) buffer tablets.

The test operation is structured as follows.

1. Create a standard inhibition curve using known concentrations of a-rdMAB. The curve is constructed over the a-Id9i degree range corresponding to CEAjI degrees 1 to 100 ■/d. In this step, a-IdMAB is a-IdMAB in PBS3d.
Added to CEA-PVC.

2. Add patient serum to a-CEA-PVC.

Due to the high specificity of monoclonal antibodies, extraction of protein from baits, which is required in many commercially available kits, is usually unnecessary.

3. Incubate the test tube at 45°C for 20 minutes.

4. Wash the test tube three times with PBS. This involves adding PBS3d to each tube and pouring the liquid into a waste bottle. The test tubes are then dried.

5. Add I-labeled a-Id monoclonal antibody to each test tube and incubate at 45°C for 30 minutes.

6. Wash the test tube three times with PBS and dry.

7. Count the test tubes in a gamma scintillation spectrometer.

8. Determine the CEA concentration in the patient's serum from the standard inhibition curve. That is, the amount of CEA in serum is a-CEA-P
It is related to the blocking effect on a-Id binding to VC.

There are many benefits to the above kit that improve it over what is currently on the market. This test has higher sensitivity. This is because a standard inhibition curve can be generated over a wide range of CEA concentrations by adjusting the amount of a-Id monoclonal antibody. This kit also has higher accuracy.

Because specific a-Id monoclonal antibodies are available in unlimited quantities from cloned hybridoma cultures, the assay does not vary from loft to loft. Additionally, continuous biochemical isolation of CEA is not required, thereby reducing costs and eliminating lot-to-lot variation.

Non-isotopic diagnostic kits using a-IdMAB as the antigen can also be easily constructed.

This kit is similar to the kit described above, except that peroxidase-conjugated a-IdMAB is used as a surrogate antigen instead of I-radiolabeled a-IdMABO. The procedure is the same as that described for RIA above, but with the addition of a chromogen (such as orthophenylenediamine) in the presence of H2O2;
and differs in that it involves the use of a spectrophotometer to measure CEA binding. The peroxidase linkages a-[d can be attached in a conventional manner, for example by coupling with glutaraldehyde.

The above illustration suggests that instead of anti-CEA, other tumor-associated antigens, such as alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), colon-specific antigen-p (c3
It will be appreciated that specific antibodies, preferably monoclonal antibodies, can be modified to specifically bind prostatic acid phosphatase (PAP), prostatic acid phosphatase (PAP), and the like.

Alternatively, the specific antibody may be an antibody that specifically binds a marker associated with an infectious lesion, such as an antibody against viruses, bacteria or other infectious microorganisms, hormones, enzymes, etc.

The method of the invention can be used in any assay in which an antigen is detected by blocking the reaction between an antibody and its complementary anti-idiotypic antibody.

More generally, any competitive immunoassay that detects antigen by its ability to block the formation of antigen/antibody complexes, typically either a-Id or a-/l, is transferred to a solid phase. In the combined form and the other component labeled,
It can be modified to use anti-idiotype antibodies in place of antigens.

The foregoing general description and examples are for illustrative purposes only, and those skilled in the art will recognize that various changes and modifications can be made without departing from the spirit of the invention as defined in the claims. Probably.

Patent applicant: Immunomedinx, Inc.

Claims (15)

[Claims] (1) A method for measuring the presence of an antigen (Ag) in a liquid sample, the method comprising: Incubate in the presence of a given amount of anti-idiotypic antibody (a-Id) that specifically binds an epitope on the hypervariable region of a-Ag, and inhibit the formation of the a-Ag a-Id complex. The step of measuring the degree of a-Ag and a
-Id, at least one of which is labeled; (b) a standard solution of unlabeled a-Id is used in step (a);
a-Ag and a-I labeled as in )
d and a-Ag·a-
measuring a standard value of the degree of Id complex formation, thereby determining the degree of a-Ag in steps (a) and (b).
- The aforementioned measuring method, characterized in that the presence of the antigen is determined from the relative degree of a-Id complex formation. (2) The method of item 1, wherein the a-Ag is directly or indirectly bound to a solid support, and the a-Id is labeled. (3) The method of item 2, wherein the a-Ag is covalently bonded to the solid support. (4) a sufficient amount of said a-Ag to bind all of said a-Ag used in step (a) or step (b);
3. The method of claim 2, wherein an isotype-specific antibody that specifically binds an epip on a region other than the hypervariable region of Ag is bound to the solid support. (5) The method of item 2, wherein the label is a radionuclide, an enzyme, a fluorescent dye, or a hapten that can be bound by an antibody specific thereto. (6) The method of item 1, wherein the a-Id is directly or indirectly bound to a solid support, and the a-Ag is labeled. (7) (a) fusing lymphocytes from an animal with a growing tumor capable of secreting idiotypic antibodies with myeloma cells; and (b) fusing an epitope on the hypervariable region of said idiotypic antibody isolating and cloning the resulting hybridoma that secretes a specifically binding monoclonal antibody. (8) The method of item 7, wherein the lymphocytes are splenocytes. (9) The method of item 7, wherein the animal is a mouse. (10) The method of item 7, wherein the myeloma cells are murine myeloma cells. (11) The method of item 1, wherein the antigen is a tumor-related marker. (12) The method of item 11, wherein the antigen is carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin or its beta subunit, colon-specific antigen p, or prostatic acid phosphatase. (13) The method of item 1, wherein the antigen is a marker produced by or associated with an infected lesion. (14) The method according to item 13, wherein the antigen is a virus, bacteria, fungus, or other microorganism, or an antigenic fragment thereof. (15) A kit for use in immunoassay of an antigen (Ag) in a liquid sample, comprising: (a) an antibody (a-Ag) that specifically binds an epitope on the antigen; (b) the a - an anti-idiotypic antibody (a-Id) that specifically binds an epitope on the hypervariable region of Ag; a labeled a-Id standard, at least one of said a-Ag and a-Id being labeled, and comprising means for preparing a solution of said antibody.